5,6-dihydro-4H-benzo[b]thieno-[2,3-d]azepine derivative

ABSTRACT

There is provided a 5,6-dihydro-4H-benzo[b]thieno-[2,3-d]azepine derivative which is useful in the treatment of respiratory syncytial virus (RSV) infection and for the prevention of disease associated with RSV infection. (Formula (1)).

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a National Stage Application claiming thepriority of co-pending PCT Application No. PCT/GB2015/052944, filed Oct.8, 2015, which in turn, claims priority from European Application No.14188494.0, filed Oct. 10, 2014. Applicant claims the benefits of 35U.S.C. §120 as to the PCT application and priority under 35 U.S.C. §119as to the said European application, and the entire disclosures of bothapplications are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to a novel compound, compositions containing thesame, processes for making said compound and its use in therapy. Thecompound of the invention is intended to treat or prevent respiratorysyncytial virus infections and associated disease particularlyinfections caused by the A and B strains thereof.

BACKGROUND OF THE INVENTION

Human respiratory syncytial virus (RSV) is a pneumovirus of theparamyxovirus family and the most common cause of bronchiolitis andpneumonia in infants under one year of age. Most children becomeinfected with RSV prior to their second birthday resulting in 75-125,000hospitalisations. The associated medical costs are thought to exceed$650 million annually in the United States alone. In addition,early-life respiratory viral infections, notably with RSV, increase therisk of the subsequent development of childhood asthma (Holt and Sly,2002.). RSV infection can produce severe, lower respiratory tractdisease in patients of any age. The elderly, as well as those havingcompromised cardiac, pulmonary or immune systems are particularlyvulnerable and it is estimated that some 14,000 deaths occur annually inthe United States in subjects over 65 years old. In addition, RSVinfection is increasingly regarded as an important precipitator ofexacerbations in patients suffering from chronic obstructive pulmonarydisease (COPD) (Mohan et al., 2010) as well as asthma (Newcomb andPeebles, 2009) and cystic fibrosis (Abman et al., 1988). Inimmuno-compromised adults, approximately 50% of upper respiratory tractinfections with RSV progress to pneumonia.

The initial portal of entry by RSV is through the nose or eye ratherthan the mouth (Hall et al., 1981). Once established in the upperrespiratory tract the virus is able to migrate readily into the lungs.The pathophysiology of RSV infection was investigated in a study of lungtissues obtained from deceased children (Johnson et al., 2007).Examination of tissues from four individuals revealed immunostaining ofepithelial cells indicating the presence of RSV, without basal cellsbeing affected. The epithelial localisation of the pathogenic organismprovides a challenge to treatment since a supra-effective concentrationof the drug substance has to be maintained at the discrete cellular siteto enable the infection to be treated and subsequently cleared.

The RSV virus exists as two antigenic sub-groups: A and B. Viruses ofthe RSV A strain were formerly regarded as the sub-group pathogensresponsible for the majority of clinical disease and were reported toproduce a more symptomatic pathology (Walsh et al., 1997; Panayiotou etal., 2014). A common RSV A strain is RSV A2 (Olivier et al., 2009).However, during a recent outbreak in China virus strains from the RSV Bsub-group were found to predominate in the afflicted population (Zhanget al., 2010).

Over the last two decades considerable progress has been made in thetreatment of a number of viral diseases including human immunodeficiencyvirus (HIV) and both hepatitis B and hepatitis C. In all these casesgold standard therapies have evolved that consist of combinationtreatments that were brought about, at least to some extent, in responseto the emergence of drug resistant disease.

FDA-approved drugs for the treatment of acute RSV infections comprise of(aerosolised) ribavirin and the humanized, monoclonal antibody,palivizumab (Synagis). The latter agent targets the RSV fusion (F)protein and is limited to prophylactic use in high risk paediatricpatients. Furthermore, clinical variants resistant to neutralisation bypalivizumab were recently identified (Zhu et al., 2011) and therefore notruly effective vaccine is currently available. The use of ribavirin islimited by its low potency against the virus and by concerns over itsside-effect profile. Consequently there is an urgent, unmet need for thediscovery of novel, safe and effective therapies against RSV infectionhaving an improved clinical profile. Moreover, in view of the emergingprominence of the RSV B strains in clinical disease it is highlydesirable that these treatments be efficacious against infectionsarising from both RSV A and RSV B strains.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a compound of formula (I),

which is:N-(2-fluoro-6-methylphenyl)-6-(4-(5-methyl-2-(7-oxa-2-azaspiro[3.5]nonan-2-yl)nicotinamido)benzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxamide.or a pharmaceutically acceptable salt thereof (“the compound of theinvention”).

Biological data disclosed herein reveals that the compound of theinvention inhibits the cytopathic effect associated with infection byRSV A strains, and also inhibits the cytopathic effect associated withinfection by RSV B strains.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of Compound (I) on virus titre in RSV A2infected air-liquid interface (ALI) cultured epithelial cells followingearly intervention with test compound

FIG. 2 shows the effect of Compound (I) on virus titre in RSV A2infected air-liquid interface (ALI) cultured epithelial cells followinglate intervention with test compound

FIG. 3 shows the effect of Compound (I) on virus titre in the lungs ofRSV A2 infected mice

FIG. 4 shows the effect of Compound (I) on virus titre in the lungs ofRSV A2 infected cotton rats

DETAILED DESCRIPTION OF THE INVENTION

Pharmaceutically acceptable salts of the compound of formula (I) includein particular pharmaceutically acceptable acid addition salts of thesaid compound. The pharmaceutically acceptable acid addition salts ofthe compound of formula (I) are meant to comprise the therapeuticallyactive non-toxic acid addition salts that the compound of formula (I) isable to form. These pharmaceutically acceptable acid addition salts canconveniently be obtained by treating the free base form with suchappropriate acids in a suitable solvent or mixture of solvents.Appropriate acids comprise, for example, inorganic acids such ashydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric,nitric, phosphoric acids and the like; or organic acids such as, forexample, acetic, propanoic, hydroxyacetic, lactic, pyruvic, malonic,succinic, maleic, fumaric, malic, tartaric, citric, methanesulfonic,ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic,p-aminosalicylic, pamoic acid and the like.

Conversely said salt forms can be converted by treatment with anappropriate base into the free base form.

Salts as referred to herein, for example in relation to intermediatecompounds, include pharmaceutically acceptable salts, such as thoseabove mentioned, as well as other salts that may be disfavoured forpharmaceutical use. Salts of acidic compounds include salts formed withpositive ions of Group 1 and Group 2 metals including, sodium,potassium, calcium and magnesium ions as well as with inorganic cationssuch as ammonium ion.

The definition of the compound of formula (I) is intended to include allstereoisomers of said compound. Stereoisomers as employed herein refersto isomeric molecules that have the same molecular formula and sequenceof bonded atoms (constitution), but that differ only in thethree-dimensional orientations of their atoms in space. This contrastswith structural isomers, which share the same molecular formula, but thebond connections and/or their order differ(s) between differentatoms/groups. In stereoisomers, the order and bond connections of theconstituent atoms remain the same, but their orientation in spacediffers.

The definition of the compound of formula (I) is intended to include alltautomers of said compound.

The definition of the compound of formula (I) is intended to include allsolvates of said compound (including solvates of salts of said compound)unless the context specifically indicates otherwise. Examples ofsolvates include hydrates.

The compound of the disclosure includes isotopic variants in which oneor more specified atom(s) is/are naturally occurring or non-naturallyoccurring isotopes. In one embodiment the isotope is a stable isotope.Thus the compound of the disclosure includes, for example deuteriumlabelled versions and the like.

The disclosure also extends to all polymorphic forms of the compoundherein defined.

Novel intermediates as described herein [such as, for example, compoundsof formula (II), (III), (IV), (VI) and (X)] form a further aspect of theinvention, as do salts thereof (such as pharmaceutically acceptablesalts).

The compound of the invention is useful as a pharmaceutical.

In an embodiment there is provided a pharmaceutical compositioncomprising the compound of the invention optionally in combination withone or more pharmaceutically acceptable diluents or carriers.

Suitably the compound of the invention is administered topically to thelung or nose, particularly, topically to the lung. Thus, in anembodiment there is provided a pharmaceutical composition comprising thecompound of the invention optionally in combination with one or moretopically acceptable diluents or carriers.

Suitably compositions for pulmonary or intranasal administration includepowders, liquid solutions, liquid suspensions, nasal drops comprisingsolutions or suspensions or pressurised or non-pressurised aerosols.

The compositions may conveniently be administered in unit dosage formand may be prepared by any of the methods well-known in thepharmaceutical art, for example as described in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,(1985). The compositions may also conveniently be administered inmultiple unit dosage form.

Topical administration to the nose or lung may be achieved by use of anon-pressurised formulation such as an aqueous solution or suspension.Such formulations may be administered by means of a nebuliser e.g. onethat can be hand-held and portable or for home or hospital use (i.e.non-portable). An example device is a RESPIMAT inhaler. The formulationmay comprise excipients such as water, buffers, tonicity adjustingagents, pH adjusting agents, viscosity modifiers, surfactants andco-solvents (such as ethanol). Suspension liquid and aerosolformulations (whether pressurised or unpressurised) will typicallycontain the compound of the invention in finely divided form, forexample with a D₅₀ of 0.5-10 μm e.g. around 1-5 μm. Particle sizedistributions may be represented using D₁₀, D₅₀ and D₉₀ values. The D₅₀median value of particle size distributions is defined as the particlesize in microns that divides the distribution in half. The measurementderived from laser diffraction is more accurately described as a volumedistribution, and consequently the D₅₀ value obtained using thisprocedure is more meaningfully referred to as a Dv₅₀ value (median for avolume distribution). As used herein Dv values refer to particle sizedistributions measured using laser diffraction. Similarly, D₁₀ and D₉₀values, used in the context of laser diffraction, are taken to mean Dv₁₀and Dv₉₀ values and refer to the particle size whereby 10% of thedistribution lies below the D₁₀ value, and 90% of the distribution liesbelow the D₉₀ value, respectively.

According to one specific aspect of the invention there is provided apharmaceutical composition comprising the compound of the invention inparticulate form suspended in an aqueous medium. The aqueous mediumtypically comprises water and one or more excipients selected frombuffers, tonicity adjusting agents, pH adjusting agents, viscositymodifiers and surfactants.

Topical administration to the nose or lung may also be achieved by useof an aerosol formulation. Aerosol formulations typically comprise theactive ingredient suspended or dissolved in a suitable aerosolpropellant, such as a chlorofluorocarbon (CFC) or a hydrofluorocarbon(HFC). Suitable CFC propellants include trichloromonofluoromethane(propellant 11), dichlorotetrafluoromethane (propellant 114), anddichlorodifluoromethane (propellant 12). Suitable HFC propellantsinclude tetrafluoroethane (HFC-134a) and heptafluoropropane (HFC-227).The propellant typically comprises 40%-99.5% e.g. 40%-90% by weight ofthe total inhalation composition. The formulation may compriseexcipients including co-solvents (e.g. ethanol) and surfactants (e.g.lecithin, sorbitan trioleate and the like). Other possible excipientsinclude polyethylene glycol, polyvinylpyrrolidone, glycerine and thelike. Aerosol formulations are packaged in canisters and a suitable doseis delivered by means of a metering valve (e.g. as supplied by Bespak,Valois or 3M or alternatively by Aptar, Coster or Vari).

Topical administration to the lung may also be achieved by use of adry-powder formulation. A dry powder formulation will contain thecompound of the disclosure in finely divided form, typically with an MMDof 1-10 μm or a D₅₀ of 0.5-10 μm e.g. around 1-5 μm. Powders of thecompound of the invention in finely divided form may be prepared by amicronisation process or similar size reduction process. Micronisationmay be performed using a jet mill such as those manufactured by HosokawaAlpine. The resultant particle size distribution may be measured usinglaser diffraction (e.g. with a Malvern Mastersizer 2000S instrument).The formulation will typically contain a topically acceptable diluentsuch as lactose, glucose or mannitol (preferably lactose), usually ofcomparatively large particle size e.g. an MMD of 50 μm or more, e.g. 100μm or more or a D₅₀ of 40-150 μm. As used herein, the term “lactose”refers to a lactose-containing component, including α-lactosemonohydrate, β-lactose monohydrate, α-lactose anhydrous, β-lactoseanhydrous and amorphous lactose. Lactose components may be processed bymicronisation, sieving, milling, compression, agglomeration or spraydrying. Commercially available forms of lactose in various forms arealso encompassed, for example Lactohale® (inhalation grade lactose; DFEPharma), InhaLac®70 (sieved lactose for dry powder inhaler; Meggle),Pharmatose® (DFE Pharma) and Respitose® (sieved inhalation gradelactose; DFE Pharma) products. In one embodiment, the lactose componentis selected from the group consisting of α-lactose monohydrate,α-lactose anhydrous and amorphous lactose. Preferably, the lactose isα-lactose monohydrate.

Dry powder formulations may also contain other excipients such as sodiumstearate, calcium stearate or magnesium stearate.

A dry powder formulation is typically delivered using a dry powderinhaler (DPI) device. Example dry powder delivery systems includeSPINHALER, DISKHALER, TURBOHALER, DISKUS, SKYEHALER, ACCUHALER andCLICKHALER. Further examples of dry powder delivery systems includeECLIPSE, NEXT, ROTAHALER, HANDIHALER, AEROLISER, CYCLOHALER,BREEZHALER/NEOHALER, MONODOSE, FLOWCAPS, TWINCAPS, X-CAPS, TURBOSPIN,ELPENHALER, MIATHALER, TWISTHALER, NOVOLIZER, PRESSAIR, ELLIPTA, ORIELdry powder inhaler, MICRODOSE, PULVINAL, EASYHALER, ULTRAHALER, TAIFUN,PULMOJET, OMNIHALER, GYROHALER, TAPER, CONIX, XCELOVAIR and PROHALER.

The compound of the invention is useful in the treatment of RSVinfection and for the prevention or treatment of disease associated withRSV infection.

In an aspect of the invention there is provided use of the compound ofthe invention for the manufacture of a medicament for the treatment ofRSV infection and for the prevention or treatment of disease associatedwith RSV infection.

In another aspect of the invention there is provided a method oftreatment of a subject infected with RSV which comprises administeringto said subject an effective amount of the compound of the invention.

In another aspect of the invention there is provided a method ofprevention or treatment of disease associated with RSV infection in asubject which comprises administering to said subject an effectiveamount of the compound of the invention.

The compound of the invention may be used in a prophylactic setting byits administration prior to infection.

In one embodiment the RSV infection is RSV A strain infection (e.g. withan RSV A2 strain). In another embodiment the RSV infection is RSV Bstrain infection (e.g. with RSV B Washington strain).

Subjects include human and animal subjects, especially human subjects.

The compound of the invention is especially useful for the treatment ofRSV infection and for the prevention or treatment of disease associatedwith RSV infection in at risk subjects. At risk subjects includepremature infants, children with congenital defects of the lung orheart, immunocompromised subjects (e.g. those suffering from HIVinfection), elderly subjects and subjects suffering from a chronichealth condition affecting the heart or lung (e.g. congestive heartfailure or chronic obstructive pulmonary disease).

The compound of the invention may be administered in combination with asecond or further active ingredient. The compound of the invention maybe co-formulated with a second or further active ingredient or thesecond or further active ingredient may be formulated to be administeredseparately by the same or a different route. According to an aspect ofthe invention there is provided a kit of parts comprising (a) apharmaceutical composition comprising the compound of the inventionoptionally in combination with one or more diluents or carriers; (b) apharmaceutical composition comprising a second active ingredientoptionally in combination with one or more diluents or carriers; (c)optionally one or more further pharmaceutical compositions eachcomprising a third or further active ingredient optionally incombination with one or more diluents or carriers; and (d) instructionsfor the administration of the pharmaceutical compositions to a subjectin need thereof. The subject in need thereof may suffer from or besusceptible to RSV infection.

Second or further active ingredients include active ingredients suitablefor the treatment or prevention of RSV infection or disease associatedwith RSV infection or conditions co-morbid with RSV infection.

Second or further active ingredients may, for example, be selected fromanti-viral agents (such as other anti-RSV agents) including F proteininhibitors (including anti-F-protein antibodies, such as palivizumab),RNA polymerase inhibitors and ribavirin and anti-inflammatory agents.

The compound of the invention may be administered at a suitableinterval, for example once per day, twice per day, three times per dayor four times per day.

A suitable dose amount for a human of average weight (50-70 kg) isexpected to be around 50 μg to 10 mg/day e.g. 500 μg to 5 mg/dayalthough the precise dose to be administered may be determined by askilled person.

The compound of the invention is expected to have one or more of thefollowing favourable attributes:

potent inhibition of cytopathic effect and/or virus replication and/orF-protein expression in humans (or an animal model, or an in vitrosystem) caused by RSV A strains, such as the A2 strain;

potent inhibition of cytopathic effect and/or virus replication and/orF-protein expression in humans (or an animal model, or an in vitrosystem) caused by RSV B strains;

long duration of action in lungs, preferably consistent with once dailydosing; and

acceptable safety profile, especially following topical administrationto the lung or nose.

Experimental Section

Abbreviations used herein are defined below (Table 1). Any abbreviationsnot defined are intended to convey their generally accepted meaning.

Table 1: Abbreviations

-   -   ALI air liquid interface    -   aq aqueous    -   BALF bronchoalveolar lavage fluid    -   BEAS2B SV40-immortalised human bronchial epithelial cell line    -   br broad    -   BSA bovine serum albumin    -   CC₅₀ 50% cell cytotoxicity concentration    -   conc concentrated    -   CPE cytopathic effect    -   d doublet    -   DAB 3,3′-diaminobenzidine    -   DCM dichloromethane    -   DMEM Dulbecco's Modified Eagle Medium    -   DMF N,N-dimethylformamide    -   DMSO dimethyl sulfoxide    -   DSS dextran sodium sulphate    -   (ES⁺) electrospray ionization, positive mode    -   Et ethyl    -   EtOAc ethyl acetate    -   FBS foetal bovine serum    -   Hep2 human laryngeal epithelioma cell line 2    -   HPLC reverse phase high performance liquid chromatography    -   hr hour(s)    -   HRP horse radish peroxidase    -   IC₅₀ 50% inhibitory concentration    -   IC₇₅ 75% inhibitory concentration    -   IC₉₀ 90% inhibitory concentration    -   IgG immunoglobin G    -   m multiplet    -   (M+H)⁺ protonated molecular ion    -   Me methyl    -   MHz megahertz    -   MMD mass median diameter    -   MOI multiplicity of infection    -   min minute(s)    -   m/z mass-to-charge ratio    -   NMP N-methylpyrrolidine    -   NMR nuclear magnetic resonance (spectroscopy)    -   nt not tested    -   OD optical density    -   PBS phosphate buffered saline    -   PCR polymerase chain reaction    -   Pen Srep Penicillin-Streptomycin    -   PFU plaque forming unit    -   prep HPLC preparative high performance liquid chromatography    -   q quartet    -   RT room temperature    -   RPMI Roswell Park Memorial Institute medium    -   RSV respiratory syncytial virus    -   s singlet    -   sat saturated    -   SDS sodium dodecyl sulphate    -   t triplet    -   THF tetrahydrofuran    -   TLC thin layer chromatography    -   TMB 3,3′,5,5′-tetramethylbenzidine    -   vol volume(s)    -   WB washing buffer        General Procedures

All starting materials and solvents were obtained either from commercialsources or prepared according to the literature citation. Unlessotherwise stated all reactions were stirred. Organic solutions wereroutinely dried over anhydrous magnesium sulfate. Hydrogenations wereperformed on a Thales H-cube flow reactor under the conditions stated.

Column chromatography was performed on pre-packed silica (230-400 mesh,40-63 μm) cartridges using the amount indicated. SCX was purchased fromSupelco and treated with 1M hydrochloric acid prior to use. Unlessstated otherwise the reaction mixture to be purified was first dilutedwith MeOH and made acidic with a few drops of AcOH. This solution wasloaded directly onto the SCX and washed with MeOH. The desired materialwas then eluted by washing with 0.7 M NH₃ in MeOH.

Preparative Reverse Phase High Performance Liquid Chromatography:

Waters X-Select CSH column C18, 5 μm (19×50 mm), flow rate 28 mL min⁻¹eluting with a H₂O-MeCN gradient containing 0.1% v/v formic acid over6.5 min using UV detection at 254 nm. Gradient information: 0.0-0.2 min,35% MeCN; 0.2-5.5 min, ramped from 35% MeCN to 65% MeCN; 5.5-5.6 min,ramped from 65% MeCN to 95% MeCN; 5.6-6.5 min, held at 95% MeCN.

Analytical and Spectroscopic Methods

Reverse Phase HPLC Conditions for LCMS Analysis:

Waters Xselect CSH C18 XP column, 2.5 μm (4.6×30 mm) at 40° C.; flowrate 2.5-4.5 mL min⁻¹ eluted with a H₂O-MeCN gradient containing 0.1%v/v formic acid over 4 min employing UV detection at 254 nm. Gradientinformation: 0-3.00 min, ramped from 95% H₂O-5% MeCN to 5% H₂O-95% MeCN;3.00-3.01 min, held at 5% H₂O-95% MeCN, flow rate increased to 4.5 mLmin⁻¹; 3.01 3.50 min, held at 5% H₂O-95% MeCN; 3.50-3.60 min, returnedto 95% H₂O-5% MeCN, flow rate reduced to 3.50 mL min⁻¹; 3.60-3.90 min,held at 95% H₂O-5% MeCN; 3.90-4.00 min, held at 95% H₂O-5% MeCN, flowrate reduced to 2.5 mL min⁻¹.

¹H NMR Spectroscopy:

Spectra were acquired on a Bruker Avance III spectrometer at 400 MHzusing residual undeuterated solvent as reference and unless specifiedotherwise were run in DMSO-d₆.

Methods for the Synthesis of Compound (I)

Non limiting synthetic strategies which have been used to prepare thecompound of the present invention are summarised below (Scheme 1). Allof the disclosed routes to Compound (I) originate from the azepinederivative, intermediate (VIIIa), which is readily accessible, in threesteps, from commercially available starting materials. The principalvariations arise from the order in which the synthetic transformationsare applied to the said key intermediate (VIIIa) thereby generatingthree different precursors to compound (I): namely Intermediates (II),(III) and (IV). Route 1, comprises of the amide coupling of thethiophene carboxylic acid (II) with 2-fluoro-6-methylaniline. Analternative preparative method, Route 2, exploits the formation ofcompound (I) from the 2-chloronicotinamide intermediate (III) by anS_(N)Ar displacement reaction with the spirocyclic amine:7-oxa-2-azaspiro[3.5]nonane. This procedure has been scaled up toprovide compound (I) in a single batch of over 0.5 kg and this syntheticcampaign is also described herein below. A third approach to compound(I) is Route 3, which consists of an amide coupling reaction between theaniline (IV) and the pre-formed 2-aminonicotinic acid (VI).

The generic groups LG and LG¹ in Scheme 1 represent leaving groups, suchthat the resulting compounds are converted into reactive electrophiles.Examples of suitable leaving groups include halogen atoms such as Cl andBr, in which Cl is typically preferred due to the ready availability anduse of reagents for their formation. Those skilled in the art willappreciate that further examples of common leaving groups, used in thiscontext, include mesylate, tosylate or triflate[p-trifluomethylsulfonate]). A review of methodologies for thepreparation of amides is covered in: ‘Amide bond formation and peptidecoupling’ Montalbetti, C. A. G. N. and Falque, V. Tetrahedron, 2005, 61,10827-10852. In the present case the alkyl group R^(a) is ethyl and,more generally, is lower alkyl such as C₁₋₆alkyl or C₁₋₄alkyl.

Ethyl 2-chloro-5-methylnicotinate

To a solution of 2-chloro-5-methylnicotinic acid (3.90 g, 22.7 mmol) inDCM (100 mL) was added oxalyl chloride (9.95 mL, 114 mmol) followed by 1drop of DMF. The resulting mixture was stirred at RT for 30 min andevaporated in vacuo. The residue thus obtained was taken up into EtOH(66 mL), stirred for a further 2 hr and then evaporated in vacuo. Thecrude product obtained was purified by flash column chromatography(SiO₂, 120 g, 0-50% DCM in isohexane, gradient elution) to afford thetitle compound as a colourless oil (3.71 g, 82% yield); ¹H NMR δ: 1.32(3H, t), 2.34 (3H, s), 4.34 (2H, q), 8.06-8.07 (1H, m), 8.41-8.43 (1H,m). [See also: Yamamoto S. et al., Bioorg. Med. Chem. 2012, 20,422-434.]

Ethyl 5-methyl-2-(7-oxa-2-azaspiro[3.5]nonan-2-yl)nicotinate

A mixture of ethyl 2-chloro-5-methylnicotinate (3.70 g, 18.5 mmol),7-oxa-2-azaspiro[3.5] nonane hemi oxalate (9.57 g, 55.6 mmol) and DIPEA(19.4 mL, 111 mmol) in NMP (50 mL) was heated at 150° C. for 2 hr. Aftercooling to RT the crude mixture was poured into water (200 mL) andextracted with EtOAc (3×200 mL). The combined organic extracts werewashed with brine (2×100 mL), and then dried and evaporated in vacuo toafford the title compound (4.81 g, g, 88% yield); R^(t) 1.32 min; m/z291 (M+H)⁺ (ES⁺); ¹H NMR δ: 1.29 (3H, t), 1.67 (4H, br t), 2.18 (3H, s),3.52 (4H, br t), 3.67 (4H, s), 4.25 (2H, q), 7.74 (1H, apparent dd),8.12 (1H, apparent dd).

5-Methyl-2-(7-oxa-2-azaspiro[3.5]nonan-2-yl)nicotinic acid: Intermediate(VI)

A mixture of ethyl5-methyl-2-(7-oxa-2-azaspiro[3.5]nonan-2-yl)nicotinate (4.1 g, 14 mmol)and lithium hydroxide (0.50 g, 21 mmol) in THF:water (4:1, 50 mL) washeated at 50° C. for 18 hr and then evaporated in vacuo. The residuethus obtained was acidified to pH 4 by the addition of 1 M hydrochloricacid and the resulting mixture extracted with EtOAc (10×250 mL). Thecombined organic extracts were evaporated in vacuo to afford the titlecompound as a crystalline solid (3.4 g, 92% yield); R^(t) 0.42 min; m/z263 (M+H)⁺ (ES⁺); ¹H NMR δ: 1.67 (4H, br t), 2.17 (3H, s), 3.52 (4H, brt), 3.69 (4H, s), 7.74 (1H, apparent dd), 8.09 (1H, apparent dd), 12.69(1H, br).

1-(4-Nitrobenzoyl)-1,2,3,4-tetrahydro-5H-benzo[b]azepin-5-one

To a solution of 1,2,3,4-tetrahydro-benzo[b]azepin-5-one (25.0 g, 155mmol) in pyridine (124 mL) at RT was added dropwise a solution of4-nitrobenzoyl chloride (57.6 g, 310 mmol) in MeCN (124 mL). Theresulting mixture was stirred at RT for 16 hr and was then quenchedcarefully with water (50 mL) and extracted with EtOAc (100 mL). Theorganic extracts were washed sequentially with sat aq NaHCO₃ (100 mL),sat aq NH₄Cl (2×100 mL), water (100 mL), brine (100 mL), and finallywith 1 M hydrochloric acid (2×100 mL), dried and the volatilesevaporated in vacuo. The crude solid thus obtained was slurried withMeOH (300 mL) and was collected by filtration and dried to afford thetitle compound as a light yellow solid (44.8 g, 93% pure by HPLC, 93%yield); R^(t) 1.92 min; m/z 311 (M+H)⁺ (ES⁺). This material was used inthe subsequent step without additional purification.

5-Chloro-1-(4-nitrobenzoyl)-2,3-dihydro-1H-benzo[b]azepine-4-carbaldehyde

To neat DMF (236 mL) at 0° C. was added dropwise phosphoryl trichloride(15.8 mL, 170 mmol) and the resulting mixture treated with a solution of1-(4-nitrobenzoyl)-1,2,3,4-tetrahydro-5H-benzo[b]azepin-5-one (44.8 g.141 mmol) in DMF (141 mL) [the latter obtained by heating a suspensionat 90° C. until full dissolution of the solid had occurred and thesolution added whilst still hot] whilst maintaining the internal tempbetween 0-5° C. The reaction mixture was stirred at 0° C. for 15 min,then allowed to attain RT for 30 min and afterwards was heated at 80° C.for 72 hr. The resulting mixture was cooled to RT and was partitionedbetween EtOAc (500 mL) and sat aq NaOAc (500 mL). The aq layer wasseparated and was washed with EtOAc (2×500 mL). The combined organicextracts were washed with brine (8×300 mL), and then dried andevaporated in vacuo to give a brown solid. The crude product thusobtained was slurried with MeOH (300 mL) and was collected by filtrationand dried to afford the title compound as a yellow solid (25.8 g, 88%pure by HPLC, 51% yield); R^(t) 2.28 min; m/z 357 (M+H)⁺ (ES⁺). Thismaterial was used in the subsequent step without additionalpurification.

Ethyl6-(4-nitrobenzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxylate:Intermediate (VIIIa)

To a solution of5-chloro-1-(4-nitrobenzoyl)-2,3-dihydro-1H-benzo[b]azepine-4-carbaldehyde(36.6 g, 89.0 mmol) in pyridine (260 mL) at RT was added ethyl2-mercaptoacetate (18.6 mL, 170 mmol) followed by triethylamine (81.0mL). The reaction mixture was heated at 70° C. for 1 hr, and at 118° C.for 2 hr and was then cooled to RT. The white precipitate that formedwas removed by filtration and the filtrate concentrated in vacuo. Theresulting residue was taken up in DCM (100 mL) and was washed with water(100 mL) and then with 1 M hydrochloric acid (70 mL). The organicextracts were dried and evaporated in vacuo. The crude solid thusobtained was slurried with MeOH (150 mL), collected by filtration anddried to afford the title compound as a yellow solid (34.2 g, 84%yield); R^(t) 2.65 min; m/z 423 (M+H)⁺ (ES⁺); ¹H NMR δ: 1.32 (3H, t),3.09-3.17 (1H, m), 3.28-3.41 (assume 2H, obscured by solvent), 4.33 (2H,q), 4.83-4.92 (1H, m), 6.96 (1H, br d), 7.10 (1H, td), 7.24 (2H, br d),7.28 (1H, td), 7.78-7.81 (2H, over-lapping s and dd), 8.06 (2H, br d).

6-(4-Nitrobenzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxylicacid: Intermediate (VII)

To a solution of ethyl6-(4-nitrobenzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxylate(1.50 g, 3.55 mmol) in a mixture of THF:MeOH (1:1, 36 mL) was added 2 Maq NaOH (9.0 mL) and the mixture heated at 50° C. for 2 hr. Aftercooling to RT the mixture was partitioned between EtOAc (200 mL) andwater (200 mL). The aq layer was separated and was acidified to pH 3 bythe addition of 1 M hydrochloric acid and then extracted with EtOAc(2×150 mL). Removal of the volatiles in vacuo afforded the titlecompound, as a yellow solid (1.44 g, 99% yield); R^(t) 2.24 min; m/z 395(M+H)⁺ (ES⁺); ¹H NMR δ: 3.06-3.17 (1H, m), 3.27-3.40 (assume 2H,obscured by solvent), 4.83-4.92 (1H, m), 6.95 (1H, br d), 7.08 (1H, brt), 7.23-7.30 (3H, over-lapping br d and br t), 7.69 (1H, s), 7.78 (1H,dd), 8.06 (2H, br d), 13.33 (1H, br s).

Ethyl6-(4-aminobenzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxylate:Intermediate (IXa)

Catalytic Reduction Method

A solution of ethyl6-(4-nitrobenzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxylate(1.00 g, 2.37 mmol) in a mixture of THF:EtOH (1:1, 100 mL) and 1 Mhydrochloric acid (2.00 mL) was passed through a Thales H-cube (1.0mL·min⁻¹, 25° C., 55 mm 10% Pd/C Cat-Cart, full hydrogen mode). Thevolatiles were removed in vacuo to afford the title compound (0.98 g,˜100% yield); R^(t) 2.28 min; m/z 393 (M+H)⁺ (ES⁺). This material wasused in the subsequent step without additional purification.

Dissolving Metal Reduction Method

To a suspension of iron powder (5.29 g, 94.7 mmol) and ethyl6-(4-nitrobenzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxylate(8.00 g, 18.9 mmol) in IPA (80 mL) was added sat aq ammonium chloride(8.0 mL). The resulting mixture was stirred at 80° C. for 1 hr and wasthen filtered through celite. The celite pad was washed with MeOH (1.5L) and combined filtrates were evaporated in vacuo. The resultingresidue was triturated with water (400 mL) and with diethyl ether (400mL) and was dried in vacuo to afford the title compound as a yellowsolid (5.89 g, 88% pure by HPLC, 70% yield); R^(t) 2.21 min; m/z 393(M+H)⁺ (ES⁺). This material was used in subsequent steps withoutadditional purification.

Ethyl6-(4-(2-chloro-5-methylnicotinamido)benzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxylate:Intermediate (Xa)

To a suspension of 2-chloro-5-methylnicotinic acid (2.49 g, 14.5 mmol)in DCM (50 mL) was added oxalyl chloride (4.24 mL, 48.4 mmol) and onedrop of DMF. The resulting mixture was stirred at RT for 1 hr and wasthen evaporated in vacuo. The residue was taken up into DCM (25 mL) andadded to a solution of ethyl 6-(4-aminobenzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxylate (3.80 g, 9.68 mmol) in pyridine (20mL) at RT. The reaction mixture was maintained at RT for 1 hr and thenquenched by the addition of water (100 mL) and extracted with EtOAc (100mL). The aq layer was separated and was washed with EtOAc (2×100 mL).The combined organic extracts were washed with water (100 mL),evaporated in vacuo and the resulting solid triturated with water (200mL). This sequence was repeated on the same scale to afford the titlecompound as a pale yellow solid (10.0 g, 89% pure by HPLC, 95% yield);R^(t) 2.51 min; m/z 545/547 (M+H)⁺ (ES⁺). This material was used insubsequent steps without additional purification.

N-(2-fluoro-6-methylphenyl)-6-(4-nitrobenzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxamide

To a suspension of6-(4-nitrobenzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxylicacid (10.0 g, 25.4 mmol) in DCM (250 mL) was added oxalyl chloride (11.1mL, 127 mmol) followed by 1 drop of DMF. The resulting mixture wasstirred at RT for 1 hr and was then evaporated in vacuo. The residuethus obtained was taken up into DCM (100 mL) and to this solution wasadded a solution of 2-fluoro-6-methylaniline (6.35 g, 50.7 mmol) inpyridine (100 mL). The mixture was stirred at RT for 1 hr and was thenevaporated in vacuo. The residue was taken up into EtOAc (500 mL) andthe solution was washed with 1 M hydrochloric acid (2×100 mL), followedby sat aq NaHCO₃ (100 mL) and then dried and evaporated in vacuo. Thissame procedure was repeated three times with additional6-(4-nitrobenzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxylicacid (12.0 g, 30.4 mmol) to afford the title compound as a pale yellowsolid (51.1 g, 93% pure by HPLC, 87% yield); R^(t) 2.46 min; m/z 502(M+H)⁺ (ES⁺). This material was used in the subsequent step withoutadditional purification.

Ethyl6-(4-(5-methyl-2-(7-oxa-2-azaspiro[3.5]nonan-2-yl)nicotinamido)benzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxylateMethod 1: Acylation of Aniline (IXa) with 2-aminonicotinc Acid (VI)

To a suspension of 5-methyl-2-(7-oxa-2-azaspiro[3.5]nonan-2-yl)nicotinicacid (2.21 g, 8.41 mmol) in DCM (50 mL) was added oxalyl chloride (0.80mL, 9.17 mmol) followed by 1 drop of DMF. The resulting mixture wasstirred at RT for 1 hr and then a second portion of oxalyl chloride(0.80 mL, 9.17 mmol) and of DMF (1 drop) were added. After a further 30min the mixture was evaporated in vacuo and the residue thus obtainedtaken up into DCM (50 mL) and added to a solution of ethyl6-(4-aminobenzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxylate(3.00 g, 7.64 mmol) in pyridine (20 mL). The resulting mixture wasstirred at RT for 1 hr, then diluted with water (100 mL) and passedthrough a phase separator. The organic phase was evaporated in vacuo andthe residue obtained purified by flash column chromatography (SiO₂, 80g, 0-100% EtOAc in isohexane, gradient elution). The pale orange residuethat was isolated was triturated with acetonitrile (2×20 mL) and thesolid that formed was collected by filtration and dried to afford thetitle compound as a white solid (2.78 g, 94% pure by HPLC, 57% yield);R^(t) 1.95 min; m/z 637 (M+H)⁺ (ES⁺); ¹H NMR δ: 1.32 (3H, t), 1.62 (4H,br t), 2.16 (3H, s), 3.06-3.38 (assume 3H, obscured by solvent), 3.45(4H, br t), 3.60 (4H, s), 4.32 (2H, q), 4.85-4.95 (1H, br), 6.88 (1H, brd), 6.99 (2H, br d), 7.14 (1H, br t), 7.29 (1H, td), 7.45-7.53 (3H,over-lapping m), 7.79 (1H, s), 7.82 (1H, dd), 8.04 (1H, apparent dd),10.37 (1H, s).

Method 2: Displacement of 2-halonicotinamide (Xa) with a7-oxa-2-azaspiro[3.5]nonane

A suspension of ethyl6-(4-(2-chloro-5-methylnicotinamido)benzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxylate(4.97 g, 9.10 mmol) and 7-oxa-2-azaspiro[3.5] nonane hemi oxalate (5.93g, 27.3 mmol) in NMP (23 mL) and Et₃N (7.61 mL, 54.6 mmol) was heated at150° C. for 7.5 hr and then cooled to RT and left to stand for 60 hr.Water (400 mL) was added and the resulting precipitate was collected byfiltration. The solid thus obtained was purified by flash columnchromatography (SiO₂, 120 g, 0-30% THF in DCM, gradient elution) toafford the title compound as a pale yellow solid (3.72 g, 64% yield);R^(t) 1.94 min; m/z 637 (M+H)⁺ (ES⁺).

6-(4-(5-Methyl-2-(7-oxa-2-azaspiro[3.5]nonan-2-yl)nicotinamido)benzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxylicacid: Intermediate (II)

To a solution ethyl6-(4-(5-methyl-2-(7-oxa-2-azaspiro[3.5]nonan-2-yl)nicotinamido)benzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxylate(3.72 g, 5.84 mmol) in a mixture of THF:MeOH (1:1, 40 mL) was added asolution of lithium hydroxide (700 mg, 29.2 mmol) in water (40 mL). Thereaction mixture was heated to 50° C. for 1 hr and was then cooled toRT. The volatiles were removed in vacuo and the remaining aq solutionwas diluted with water and sonicated until the resulting precipitatedissolved. This mixture was neutralised by the addition of 1 Mhydrochloric acid and the resulting solid collected by filtration anddried in vacuo to afforded the title compound as an off-white solid(3.27 g, 92% yield); R^(t) 1.64 min; m/z 609 (M+H)⁺ (ES⁺); ¹H NMR δ:1.62 (4H, br t), 2.16 (3H, s), 3.00-3.51 (assume 8H, obscured bysolvent), 3.60 (4H, s), 4.82-4.96 (1H, br), 6.86 (1H, br d), 6.99 (2H,br d), 7.11 (1H, br t), 7.28 (1H, td), 7.46-7.54 (3H, over-lapping m),7.62 (1H, s), 7.78 (1H, dd), 8.03 (1H, dd), 10.38 (1H, s).

6-(4-(2-Chloro-5-methylnicotinamido)benzoyl)-N-(2-fluoro-6-methylphenyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxamide:Intermediate (IIIa)

A solution of 2-chloronicotinoyl chloride (1.21 g, 3.36 mmol) in DCM (10mL) was added to a stirred solution of6-(4-aminobenzoyl)-N-(2-fluoro-6-methylphenyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxamide (2.00 g, 4.24 mmol) in pyridine (10mL). The reaction mixture was stirred at RT for 1 hr and was then pouredinto water (100 mL) and extracted into EtOAc (2×50 mL) The combinedorganics were evaporated in vacuo and the resulting solid was slurriedin EtOAc (50 mL) and collected by filtration. The above procedure wasrepeated three times, on an increasingly greater scale, using 5.0, 15.0and finally 18.0 g of the aniline starting material. All four batcheswere combined by dissolving them in DCM (300 mL). The solvent wasevaporated in vacuo to give the title compound as a white solid (40.3 g,75% yield); R^(t) 2.39 min; m/z 625 (M+H)⁺ (ES⁺); ¹H NMR δ: 2.26 (3H,s), 2.32 (3H, s), 3.09-3.33 (assume 3H, obscured by solvent), 4.83-5.03(1H, m), 6.86 (1H, d), 7.04 (2H, d), 7.09-7.19 (3H, m), 7.22-7.34 (2H,m), 7.51 (2H, d), 7.83 (1H, dd), 7.91 (1H, d), 7.96 (1H, s), 8.36 (1H,dd), 10.04 (1H, s), 10.71 (1H, s).

6-(4-aminobenzoyl)-N-(2-fluoro-6-methylphenyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxamide:Intermediate (IV)

Method 1: Dissolving Metal Reduction

To a solution ofN-(2-fluoro-6-methylphenyl)-6-(4-nitrobenzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxamide (5.00 g, 9.97 mmol) in EtOH (100 mL)was added ammonium chloride (5.33 g, 100 mmol), water (20 mL) and theniron powder (2.78 g, 49.8 mmol). The resulting mixture was stirred atreflux for 1 hr and was then filtered through celite. The celite pad waswashed with EtOH (50 mL) and the combined filtrates were evaporated invacuo. The resulting residue was taken up into EtOAc (200 mL), washedwith water (2×100 mL) and was then dried and evaporated in vacuo. Thisprocedure was repeated three times with additionalN-(2-fluoro-6-methylphenyl)-6-(4-nitrobenzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxamide (15.0 g, 29.9 mmol) and the solids thatwere obtained were combined and triturated with Et₂O (200 mL) to affordthe title compound as a pale yellow solid (41.1 g, 87% yield); R^(t)2.12 min; m/z 472 (M+H)⁺ (ES⁺); ¹H NMR δ: 2.25 (3H, s), 3.02-3.30 (3H,br), 4.85-5.05 (1H, br), 5.51 (2H, s), 6.27 (2H, d), 6.75 (2H, d), 6.80(1H, d), 7.10-7.15 (3H, over-lapping m), 7.24-7.30 (2H, over-lapping m),7.81 (1H, dd), 7.93 (1H, s), 10.02 (1H, s).

Method 2: Catalytic Hydrogenation

To a solution ofN-(2-fluoro-6-methylphenyl)-6-(4-nitrobenzoyl)-5,6-dihydro-4Hbenzo[b]thieno[2,3-d]azepine-2-carboxamide (100 mg, 0.199 mmol) in THF (4.0 mL)was added 5% Pd/C paste (58 wt % water, 21.0 mg, 0.100 mmol) and themixture stirred under 5 bar of hydrogen for 18 hr. Upon competition ofthe reaction the mixture was passed through a Agilent 0.45 μm syringefilter and filtrate evaporated in vacuo to afford the title compound(91.0 mg, 97% yield); R^(t) 2.13 min; m/z 472 (M+H)⁺ (ES⁺).

Preparation ofN-(2-Fluoro-6-methylphenyl)-6-(4-(5-methyl-2-(7-oxa-2-azaspiro[3.5]nonan-2-yl)nicotinamido)benzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxamide:Compound (I) Route 1: Amide coupling of the thiophene carboxylic acid(II) with 2-fluoro-6-methylaniline

To a solution of6-(4-(5-methyl-2-(7-oxa-2-azaspiro[3.5]nonan-2-yl)nicotinamido)benzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxylicacid (400 mg, 0.657 mmol) in DCM (40 mL) was added1-chloro-N,N,2-trimethylprop-1-en-1-amine (174 μL, 1.31 mmol). Thereaction was stirred at RT for 1.5 hr and was then concentrated invacuo. The residue was taken up in DCM (40 mL) and an aliquot of thissolution (5.0 mL, 0.080 mmol) was added to 2-fluoro-6-methylaniline (100mg, 0.797 mmol) and the reaction mixture stirred at RT for 3 days. Thevolatiles were evaporated in vacuo and the resulting residue waspurified by preparative HPLC to afford Compound (I), as an off-whitesolid (14 mg, 23% yield); R^(t) 1.85 min; m/z 716 (M+H)⁺ (ES⁺); ¹H NMRδ: 1.62 (4H, br t), 2.17 (3H, s), 2.26 (3H, s), 3.14-3.37 (assume 3H,obscured by solvent), 3.46 (4H, br t), 3.60 (4H, s), 4.90-4.98 (1H, br),6.88 (1H, br d), 7.02 (2H, br d), 7.10-7.16 (3H, over-lapping m),7.25-7.31 (2H, over-lapping m), 7.48 (1H, d), 7.52 (2H, br d), 7.82 (1H,dd), 7.95 (1H, s), 8.04 (1H, dd), 10.03 (1H, s), 10.37 (1H, s).

Route 2: S_(N)Ar displacement of the chloronicotinamide (III) with7-oxa-2-azaspiro[3.5]nonane

To a solution of6-(4-(2-chloro-5-methylnicotinamido)benzoyl)-N-(2-fluoro-6-methylphenyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxamide(10.0 g, 16.0 mmol) and 7-oxa-2-azaspiro[3.5]nonane hemi oxalate (10.4g, 48.0 mmol) in NMP (125 mL) was added triethylamine (13 mL, 96 mmol)and the reaction mixture heated at 145° C. for 7 hr. After cooling to RTthe mixture was poured into water (800 mL) and the resulting solids werecollected by filtration, washed with water (2×100 mL) and then taken upin DCM (400 mL). The solution was washed with water (100 mL), dried oversodium sulphate and evaporated in vacuo. The solid residue was purifiedby flash column chromatography (SiO₂, 220 g, 20-100% EtOAc in diethylether, gradient elution) to afford the title compound as a white solid.This procedure was repeated on additional 5 and 10 g batches of thechloronicotinamide starting material. The three product batches werecombined by dissolution in EtOAc (500 mL) and evaporation of the solventin vacuo. The resulting solid was triturated with diethyl ether (200 mL)and the solid collected by filtration and dried to afford the titlecompound, Compound (I) as a white solid (24 g, 82% yield); R^(t) 1.88min; m/z 716 (M+H)⁺ (ES⁺).

Route 3: Amide Coupling of the Aniline (IV) with the 2-aminonicotinicAcid (VI)

To a solution of 5-methyl-2-(7-oxa-2-azaspiro[3.5]nonan-2-yl)nicotinicacid (83 mg, 0.32 mmol) in DCM (5.0 mL) was added1-chloro-N,N,2-trimethylprop-1-en-1-amine (37 μL, 0.28 mmol). Themixture was stirred at RT for 15 min and was then added to a solution of6-(4-aminobenzoyl)-N-(2-fluoro-6-methylphenyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxamide(100 mg, 0.21 mmol) in pyridine (5.0 mL). The reaction mixture wasstirred for a further 1 hr and concentrated in vacuo. The residue wastriturated with water (20 mL) and the resulting buff solid was collectedby filtration and was purified by flash column chromatography (SiO₂, 12g, 0-10% MeOH in DCM, gradient elution) to afford Compound (I), as awhite solid (55 mg, 36% yield); R^(t) 1.88 min; m/z 716 (M+H)⁺ (ES⁺).

Scale-Up of the Preparation of Compound (I) by Route 2

The synthetic methodology described above for Route 2, has beensuccessfully exploited to prepare the compound of the present inventionon a scale of >0.5 kg. Analytical and spectroscopic methods pertainingto this campaign are described below.

Analytical and Spectroscopic Methods

Reverse Phase HPLC Conditions for LCMS Analysis:

CORTECS C18⁺ 4.6×150 mm column; 2.7 μm (Ex. Waters #186007408) at 40°C.; flow rate 1.0 mL·min⁻¹ eluted with a purified H₂O-MeCN gradientcontaining 0.1% formic acid over 25 min employing UV detection at 310nm. Injection volume 5 μL. Gradient information: 0-15 min, ramped from95% H₂O-5% MeCN to 5% H₂O-95% MeCN; 15-25 min, held at 5% H₂O-95% MeCN.

¹H NMR Spectroscopy.

Spectra were acquired using a JOEL ECX 400 MHz spectrometer. Residualundeuterated solvent was used as reference and samples were run inDMSO-d₆.

1-(4-Nitrobenzoyl)-1,2,3,4-tetrahydro-5H-benzo[b]azepin-5-one

To a solution of 1,2,3,4-tetrahydrobenzazepin-5-one (2670 g, 16.6 mol)in DCM (23.2 L) was added a 30% w/v aq solution of K₂CO₃ (15.2 L).4-Nitrobenzoyl chloride (3105 g, 16.7 mol) was added portion-wise over15 min maintaining an internal temp of <25° C. The reaction was stirredat 18-25° C. for 18 hr at which point TLC (50% v/v ethyl acetate inheptane) indicated the reaction was incomplete. Additional4-nitrobenzoyl chloride (167 g, 0.9 mol) was added and the reactionstirred for a further 1.5 hr after which TLC indicated the reaction wascomplete. The phases were separated and the organics were added to asolution of 2M NaOH (10 L) and stirred for 2 hr. The phases wereseparated and the organics were washed with water (2×5 L), dried overMgSO₄ and filtered. The pad was washed with DCM (4 L) and the combinedorganics were evaporated in vacuo. The resulting solid was dried invacuo at 45° C. for 24 hr to afford the title compound as a light beigesolid (4998 g, 97% active yield; HPLC purity 96.2%, NMR purity >95%);R^(t) 10.09 min; m/z 311.1 (M+H)⁺ (ES⁺).

5-Chloro-1-(4-nitrobenzoyl)-2,3-dihydro-1H-benzo[b]azepine-4-carbaldehyde

A 50 L vessel charged with DMF (10.0 L) was cooled to 0° C. and wastreated dropwise over 1 hr with phosphoryl chloride (1802 mL, 19.33mol), whilst maintaining the internal temp below 5° C. (an exotherm from0-5° C. was observed) and was then stirred for 30 min at 0-5° C. Asolution of1-(4-nitrobenzoyl)-1,2,3,4-tetrahydro-5H-benzo[b]azepin-5-one (5000 g,16.11 mol) in DMF (10.0 L), prepared by dissolution at 70° C., was thenadded whilst warm (to avoid precipitation) to the phosphorylchloride/DMF solution via vacuum transfer over 30 min, maintaining thebatch temp between 0-10° C. On completion of the addition, the reactionwas stirred under nitrogen for 30 min at 0-5° C. and then at 80° C. for18 hr, at which time HPLC analysis showed consumption of the startingmaterial was complete. The reaction mixture was cooled to 40° C. and wasdivided into two equal portions, both of which were worked up in thesame manner, as follows. The first portion was concentrated in vacuo toapproximately half of its original volume (˜7 L) and was then added tosat aq NaOAc (34.0 L), pre-cooled to 10° C., over 2 hr (an exotherm from20-30° C. was observed). After stirring for 15 min at 20° C. the mixturewas extracted with DCM (27.2 L) and the phases were separated. The aqlayer was back-extracted with DCM (27.2 L), and the phases wereseparated. The combined organic extracts were washed with water (2×40 L)then dried over MgSO₄ (4.0 kg), filtered, and the filtrate concentrated.The same work-up procedure was repeated on the second portion of thecrude reaction mixture and combined with the first, to give the titlecompound as an oil (5119 g, 89% active yield, HPLC purity 87.6%, ¹H NMRpurity of 95%); R^(t) 11.74 min; m/z 357.2 (M+H)⁺ (ES⁺).

Ethyl6-(4-nitrobenzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxylate

To a solution of5-chloro-1-(4-nitrobenzoyl)-2,3-dihydro-1H-benzo[b]azepine-4-carbaldehyde(3418 g, 9.580 mol) in pyridine (15.83 L, 195.4 mol) at 10° C. undernitrogen was added ethyl-2-mercaptoacetate (1118 mL, 10.24 mol) dropwiseover 30 min, whilst maintaining the internal temp below 20° C. (anexotherm from 10 to 15° C. was observed). The resulting solution wasthen treated dropwise with triethylamine (7531.51 mL, 54.03 mol) over 30min, keeping the internal temp below 20° C. (no exotherm observed). Thereaction mixture was stirred at 20° C. for 1 hr and then at 70° C. for18 hr. After this time HPLC analysis revealed that consumption of thestarting chloro enal was complete and the reaction was allowed to coolto 18-25° C.

The mixture was filtered (to remove insoluble salts), and the pad waswashed with acetone (1.0 L). The combined filtrates were concentrated invacuo to remove volatiles, and the residue taken up into DCM (11964 mL)and washed with water (7623 mL). The organic phase was separated and waswashed with 1M hydrochloric acid (7623 mL) and then dried over MgSO₄.The inorganics were removed by filtration, washed with DCM (4.0 L) andthe combined filtrates were evaporated in vacuo to an oily residue.

The residue was taken up into ethanol (20500 mL) and the solution wasstirred at 60° C. for 1 hr and was then cooled to 18-25° C. and stirredat this temperature for 1 hr. The resulting solid was collected byfiltration, washed with ethanol (13.8 L) and dried at 50° C. undervacuum, to afford the title compound (2939 g, 73% active yield, HPLCpurity 96.9%, ¹H NMR purity >97%); R^(t) 13.85 min; m/z 423.2 (M+H)⁺(ES⁺).

6-(4-Nitrobenzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxylicacid

A 50 L vessel, was charged at 18-25° C. with a 1:1 mixture of THF andwater (37.63 L) and ethyl6-(4-nitrobenzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxylate(3763.7 g, 8.90 mol). To the resulting solution was added solid KOH(749.1 g, 13.35 mol) portion-wise, whilst keeping the internal tempbelow 25° C. (an exotherm from 20-22° C. was observed). The reaction washeated at 50° C. for 18 hr at which time HPLC analysis revealed thatconsumption of the starting material was complete. The reaction wasallowed to cool to 18-25° C. and the organic solvent was removed invacuo. The remaining aq solution was diluted with water (28.27 L) andthen conc hydrochloric acid (1.25 L) was added slowly to the solutionuntil pH 1 was attained (a 5° C. exotherm was observed, with moderateoff-gassing). The resulting light tan suspension was filtered and thepad was washed with water (2×9.5 L). The solid was dried in an ovenunder vacuum at 50° C. to afford the title compound (3185.5 g, 91%active yield, HPLC purity 98.0%, ¹H NMR assay 91.0%); R^(t) 11.11 min;m/z 395.2 (M+H)⁺ (ES⁺).

N-(2-Fluoro-6-methylphenyl)-6-(4-nitrobenzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxamide

A suspension in DCM (15.6 L), of6-(4-nitrobenzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxylicacid (1714 g, 1560 g active material, 3.955 mol) was placed under anitrogen atmosphere and DMF (6.2 mL, 79.1 mmol) was added to themixture. Oxalyl chloride (690 mL, 7.91 mol) was then added slowly over40 min in order to control gas evolution (an exotherm from 16.9-18.3° C.was observed) and the reaction mixture was stirred at 18-25° C.overnight. TLC analysis (8% methanol in DCM) indicated that some of thethiophene carboxylic acid starting material remained and additionaloxalyl chloride (300 mL, 3.44 mol) was added to the mixture. Afterstirring at 18-25° C. for 3 hr the reaction was complete and theresulting mixture was concentrated in vacuo to provide6-(4-nitrobenzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carbonylchloride as a dark yellow solid (¹H NMR indicated the presence of 15.4%DCM and 0.5% DMF).

The crude acid chloride so obtained was suspended in DCM (7.8 L) under anitrogen atmosphere and was treated with pyridine (480 mL, 5.93 mol).2-Fluoro-6-methylaniline (475 mL, 4.11 mol) was then added slowly over15 min, with cooling from an ice/water bath, (resulting in an exothermfrom 20.9-35.2° C.). The mixture formed a solution and was stirred at18-25° C. overnight at which point the reaction was determined to becomplete (HPLC 250 nm).

The resulting suspension was divided into two equal portions, each ofwhich were diluted with water (7.8 L), stirred for 1 hr at 18-25° C. andthen the solids collected by filtration. The two filter cakes were eachwashed with water (1.8 L) and with DCM (2×1.6 L) and combined. The solidwas dried in an oven at 50° C. to provide the title compound as anoff-white solid, (1573 g, 79% active yield, ¹H NMR purity >95%,containing 1.98% DCM and 0.56% of pyridine.HCl); R^(t) 12.76 min; m/z502.4 (M+H)⁺ (ES⁺).

6-(4-Aminobenzoyl)-N-(2-fluoro-6-methylphenyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxamide; Intermediate (IV)

To a solution ofN-(2-fluoro-6-methylphenyl)-6-(4-nitrobenzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxamide (80 g, 0.160 mol) in a mixture of DMF(240 mL) and 2-MeTHF (640 mL), under a nitrogen atmosphere, was added20% Pd(OH)₂/C catalyst (8.0 g) and the mixture sparged with hydrogen andheated to 55° C. After sparging with hydrogen for 3 hr the reaction wasplaced under a hydrogen atmosphere overnight and was then sparged withhydrogen for a further 4 h. HPLC analysis indicated the presence of98.5% product and 0.55% of a reaction intermediate (identity notconfirmed, assumed to be the nitroso or hydroxylamine intermediate).

The reaction mixture was cooled to 48° C. and was passed through aCelite pad (24 g). The Celite pad was washed with DMF (2×160 mL) and asthese washes contained catalyst they were passed through an inlinefilter. The combined filtrates were concentrated in vacuo to remove mostof the 2-MeTHF providing a DMF/product solution. This mixture was addedover 5 min to water (1.6 L) that was cooled with an ice/water bath (anexotherm was observed from 9.9-17.6° C.), providing a white suspensionwhich was stirred at 18-25° C. for 1 hr. The solids were collected byfiltration and the filter cake was washed with water (3×160 mL) and thendried in an oven at 50° C. to give the product as a white solid (75.5 g,HPLC purity 98.2%, 0.34% H₂O by KF, 6.56% DMF by ¹H NMR).

The solid so obtained was slurried in DCM (400 mL) at 18-25° C. for 65min, collected by filtration and the filter cake washed with DCM (2×160mL). This material was then oven dried at 50° C. to give the titlecompound as a white solid (72.0 g, 65.9 g active product, 88% yield,HPLC purity 98.75%; containing 8.25%, DCM and 0.19% DMF by NMR); R^(t)11.01 min; m/z 472.4 (M+H)⁺ (ES⁺).

6-(4-(2-Chloro-5-methylnicotinamido)benzoyl)-N-(2-fluoro-6-methylphenyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxamide; Intermediate (IIIa)

Oxalyl chloride (133 mL, 1.58 mol) was added to a suspension of2-chloro-5-methyl nicotinic acid (225.4 g, 1.313 mol) in DCM (2254 mL)at 18-25° C. followed by DMF (0.8 mL, 0.010 mol) (results in mildexotherm and gas evolution) and the reaction stirred at 20-25° C. for 1hr. HPLC analysis of an aliquot (quenched into methanol) indicated <1%of 2-chloro-5-methylnicotinic acid was remaining. The solvent wasremoved in vacuo and the oily residue was azeotroped with DCM (500 mL)to remove residual oxalyl chloride.

The resulting oil was taken up into DCM (413 mL) and was added dropwiseover 10 min to a suspension of6-(4-aminobenzoyl)-N-(2-fluoro-6-methylphenyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxamide (412.9 g, 0.876 mol) in a mixture ofpyridine (283 mL, 3.502 mol) and DCM (28920 mL) whilst maintaining theinternal temp <40° C. (maximum temp reached 38° C.). The reaction wasstirred at 18-25° C. for 1 hr after which time HPLC (sample quenchedinto methanol) indicated the reaction was complete (<1% of aniline s/mremaining).

Heptane (3300 mL) was added to the mixture at 18-25° C. and theresulting suspension was stirred for 15 min and the solids thencollected by filtration. The filter cake was washed with heptane (2×1650mL) and the crude solid so obtained was slurried in water (4130 mL) at90-95° C. for 30 min and then cooled to 18-25° C. The solids werecollected by filtration, washed with water (2×826 mL) and dried in avacuum oven at 50° C. to give the title compound as a white solid (504.2g, 92% active yield, HPLC purity [230 nm] 98.24%; containing 0.3%pyridine HCl by ¹H NMR and 0.4%.H₂O by KF); R^(t) 12.19 min; m/z 625.6(M+H)⁺ (ES⁺).

N-(2-Fluoro-6-methylphenyl)-6-(4-(5-methyl-2-(7-oxa-2-azaspiro[3.5]nonan-2-yl)nicotinamido)benzoyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxamide:Compound (I)

A suspension of6-(4-(2-chloro-5-methylnicotinamido)benzoyl)-N-(2-fluoro-6-methylphenyl)-5,6-dihydro-4H-benzo[b]thieno[2,3-d]azepine-2-carboxamide(563.9 g, 0.902 mol), 7-oxa-2-azaspiro[3.5]nonane hemi oxalate (233.0 g,1.353 mol) and potassium carbonate (374.0 g, 2.706 mol) in NMP (2820 mL)was stirred at 105-115° C. for 18 hr (HPLC analysis indicated theformation of 97.7% of the desired product and 0.02% ofchloro-nicotinamide remaining).

The reaction was cooled to 18-25° C. and was added to water (8459 mL)with stirring at <45° C. (mildly exothermic quench). After stirring at18-25° C. for 30 min, the resulting solid was collected by filtration,washed with water (2×1128 mL) and pulled dry. The crude product thusobtained was re-slurried in water (5640 mL) at 90-95° C. for 30 min,then cooled to 18-25° C., collected by filtration, washed with water(2×1260 mL) and dried in a vacuum oven at 50° C. to give a white solid(632.0 g).

The ¹H NMR spectrum indicated 1.7% NMP to be present and the solid wasre-slurried in water (5640 mL) at 90-95° C. for 30 min, cooled to 18-25°C., filtered, washed with water (2×1260 mL) and pulled dry. Furtherdrying in a vacuum oven at 50° C. furnished the title compound, as awhite solid (614.0 g, 95%, containing 0.75% NMP by ¹H NMR and 1.7% H₂Oby KF.) R^(t) 9.48 min; m/z 716.8 (M+H)⁺ (ES⁺); ¹H NMR δ: 1.60 (4H, brt), 2.16 (3H, s), 2.25 (3H, s), 3.14-3.30 (assume 3H, broad m, partlyobscured by solvent), 3.44 (4H, br t), 3.59 (4H, s), 4.93 (1H, br d),6.86 (1H, br d), 7.01 (2H, br d), 7.09-7.15 (3H, over-lapping m),7.24-7.30 (2H, over-lapping m), 7.48 (1H, d), 7.51 (2H, br d), 7.82 (1H,d), 7.95 (1H, s), 8.02 (1H, d), 10.04 (1H, s), 10.39 (1H, s).

Biological Testing: Experimental Methods

Assessment of RSV Induced CPE in HEp2 Cells

HEp2 cells were seeded (10³/well/50 μL) in 384-well plates (Cataloguenumber 353962, BD Falcon, Oxford, UK) in 5% serum free-DMEM containing 2mM L-glutamine and 1 mM sodium pyruvate one day before infection. RSV A2strain (#0709161v, NCPV, Public Health England, Wiltshire) or RSV BWashington strain (VR-1580, ATCC, Manassas, Va. 20108) virus solutionswere prepared in serum free-DMEM with 2 mM L-glutamine and 1 mM sodiumpyruvate, and then added (50 μL/well) to achieve a final virusconcentration of 1 MOI. Simultaneously Compound (I) (0.5 μL DMSOsolution) was added to 100 μL of HEp2 cell culture with virus solutionto provide a final DMSO solution of 0.5%. Plates were incubated (37°C./5% CO₂) for 5 days for studies using RSV A2 strain or 6 days forthose using RSV B strain, and then resazurin sodium salt (5 μL of 0.03%solution; Sigma-Aldrich, Dorset, UK) was added to each well and theplate incubated for a further 6 hr (37° C./5% CO₂). The fluorescence ofeach well [545 nm (excitation)/590 nm (emission)] was determined using amulti-scanner (Clariostar: BMG, Buckinghamshire, UK). The percentageinhibition for each well was calculated and the IC₅₀, IC₇₅ and IC₉₀values were calculated from the concentration-response curve generatedfor Compound (I).

Assessment of RSV F Protein Expression in BEAS2B Bronchial EpithelialCells

An early event which follows the infection of epithelial cells by RSV isthe expression of RSV F-protein on the cells' surface. BEAS2B cells(SV40-immortalised human bronchial epithelial cell line) were grown in96 well plates. Once more than 70% confluent, cells were infected withRSV A2 (#0709161v, NCPV, Public Health England, Wiltshire) at an MOI of0.01 in clear RPMI-1640 medium (Life technologies, Paisley, UK) with 2%FBS (Life technologies, Paisley, UK), and incubated for 3 days (37°C./5% CO₂).

Supernatant was aspirated and the cells were fixed with 4% formaldehyde(100 μL in PBS solution) for 20 min, washed 3 times with washing buffer(200 μL; PBS containing 0.05% Tween-20) and incubated with blockingsolution (100 μL; 5% Marvel milk in PBS) for 1 hr. Cells were thenwashed with washing buffer (200 μL) and incubated for 1 hr at 37° C.with anti-RSV (2F7; mouse monoclonal, lot 160290, Cat. No. ab43812,Abcam plc, Cambridge, UK) F-fusion protein antibody (50 μL; prepared ata 1:1000 dilution in 5% milk/PBS-tween). After washing, cells wereincubated with an HRP-conjugated anti-mouse IgG antibody (50 μL preparedat a 1:2000 dilution in 5% milk in PBS; lot 00095437, Cat. No. P0447,Dako UK Ltd, Cambridgeshire, UK) for 1 hr. Cells were washed twice withwashing buffer and once with PBS. TMB substrate (100 μL; substratereagent pack lot 320436, Cat. No. DY999, R&D Systems, Inc. Abingdon, UK)was then added and the reaction was stopped by the addition of aqsulfuric acid (50 μL; 2N). The resultant signal was determinedcolourimetrically (OD: 450 nm with a reference wavelength of 655 nm) ina microplate reader (Multiskan FC®, ThermoFisher Scientific). Cells werethen washed and 1% crystal violet solution (50 μL; lot SLB4576, Cat. No.HT90132-1L, Sigma-Aldrich) was applied for 30 min. After washing withPBS (200 μL) three times, 1% SDS (100 μL) was added to each well, andplates were shaken lightly for 1 hr prior to reading the absorbance at595 nm. The measured OD₄₅₀₋₆₅₅ readings were corrected for cell numberby dividing the OD₄₅₀₋₆₅₅ by the OD₅₉₅ readings. The percentageinhibition for each well was calculated and the IC₅₀ value derived fromthe concentration-response curve generated for Compound (I).

Cell Viability: Resazurin Assay

HEp2 cells were seeded in 384-well plates (10³/well/50 μL; BD Falcon Ref353962) in FBS DMEM (5%, containing 2 mM L-glutamine and 1 mM sodiumpyruvate) one day before experimentation. Serum-free DMEM (50 μL) wasadded to test wells while for control wells the media was removed andsterile water (100 μL) was added. Compound (I) (0.5 μL DMSO solution)was added to give a final DMSO concentration of 0.5%. Hep2 cells wereincubated with each test compound for 5 days (37° C./5% CO₂ in 5% FBS)and then resazurin stock solution (5 μL; 0.03%) was added to each welland the plate incubated for a further 6 hr (37° C./5% CO₂). Thefluorescence of each well at 545 nm (excitation) and 590 nm (emission)was determined using a multi-scanner (Clariostar: BMG Labtech). Thepercentage loss of cell viability was calculated for each well relativeto vehicle (0.5% DMSO) treatment.

Any apparent increase in cell viability associated with test compoundtreatment relative to vehicle is consequently tabulated as a negativepercentage. Where appropriate, a CC₅₀ value was calculated from theconcentration-response curve generated for Compound (I).

Assessment of Virus Titre in Air-Liquid Interface (ALI) CulturedBronchial Epithelial Cells

ALI cultured human bronchial epithelial cells were sourced fromEpithelix Sàrl (Geneva, Switzerland) and maintained by changing thebasal media every 3-4 days, whilst the apical surface was washed onceweekly with PBS. On day 0, the apical surface of each well was washedonce with sterile PBS (300 μL) and the inserts were transferred to new24-well plates containing fresh MucilAir culture medium (780 μL;EP04MM). RSV A2 (50 μL; diluted in MucilAir culture medium to give afinal MOI of 0.01) was added to cells for 1 hr (37° C./5% CO₂). For thepurposes of standardizing MOI calculations, each MucilAir insert wasestimated to contain 2×10⁵ apical facing cells per well. Virus inoculumwas removed with a pipette and inserts were washed twice with sterilePBS (300 μL).

Sampling was conducted by adding sterile PBS (300 μL) to the apicalsurface of each well for 5 min. The apical sample was then removed andtransferred to tubes containing 50% sucrose dissolved in PBS (100 μL)before being stored at −80° C. This harvesting procedure was repeateddaily beginning on day 0 and concluding on day 7.

ALI cultures were dosed apically with Compound (I) on days 0-7 for“early intervention” protocols, or days 3-7 for “late intervention”protocols. Compound (I) (50 μL in 0.5% DMSO/PBS) was added to the apicalsurface and incubated (37° C./5% CO₂) for 1 hr before being removed.Vehicle treatments (0.5% DMSO/PBS) were performed on the correspondingapical surfaces to ensure each well received the same number ofmanipulations. On Day 5, the basal media was removed from each well andreplenished with fresh MucilAir culture media as a necessary maintenancestep for ALI culture cells.

Virus titre was quantified by plaque assay. HEp2 cells were grown in24-well plates (Corning) for 48 hr prior to infection in DMEM containing10% FBS until they attained 100% confluency. Collected samples werethawed at RT and ten-fold serial dilutions were prepared in serum-freeDMEM. The growth medium from HEp2 cells was aspirated and replaced with300 μL of serially diluted virus collections and left to infect at 37°C./5% CO₂ for 4 hr. The infectious media was aspirated and replaced withPlaque Assay Overlay (500 μL; 1% methylcellulose in MEM, 2% FBS, 1% PenStrep, 0.5 μg/mL amphotericin B), and left for 7 days at 37° C./5% CO₂Cells were fixed with ice-cold methanol for 10 min and blocked with 5%powdered milk (Marvel) in 0.05% PBS-tween (‘blocking buffer’) for 1 hrat RT. Anti-RSV F-protein antibody (2F7; Abcam: ab43812) was diluted toa 1:100 concentration in blocking buffer and added to cells for 1 hr atRT with shaking. Cells were washed using PBS and incubated with thesecondary antibody (HRP conjugated goat anti-mouse secondary antibody(Dako P044701-2) diluted in 1:400 with blocking buffer) for 1 hr at RTwith shaking. The secondary antibody solution was removed and cells werewashed with PBS before the metal-enhanced development substrate DAB wasprepared in ultra-pure water (according to manufacturer's instructions).Each well received 300 μL of development substrate (sigmaFAST D0426)until plaques were visible. Plaques were counted by eye and confirmedusing light microscopy, allowing the calculation of plaque forming unitsper mL.

RSV Infection in Mice

Non-fasted mice (male BALB/C, 20-30 g) were infected intranasally withRSV A2 or virus diluent (DMEM, 2% FBS, 12.5% sucrose) under isoflurane(5% in O₂) anaesthesia. The A2 strain of RSV (50 uL of 1.3×10⁶ PFU/mL:final 0.65×10⁵ PFU/mouse) was instilled into each nostril in a drop wisefashion alternating between the two until a volume of 50 μL wasdelivered. Following infection each animal was weighed on a daily basisto monitor changes. Compound (I) was dissolved in 100% DMSO (at 20 mg/mLand/or 2 mg/mL), then diluted at 1:10 in isotonic saline to achieve 10%DMSO in all treatments. Formulations were then sonicated to produce asuspension. The suspension was administered intratracheally (20 μL) witha FMJ-250 PennCentury device or intranasally (40 μL) with a pipette on 1day and 1 hr before infection (day 0), and then on days 1, 2 and 3 postinfection. Four days after RSV challenge, the animals were euthanised(by intraperitoneal injection of a pentobarbitone overdose), the tracheacannulated and BALF extracted for total and differential cell counts.Following BALF collection, the right lung was removed from each animaland homogenised in ice-cold Dulbecco's modified Eagles medium (using 10times the lung weight of DMEM containing 1% BSA and 25% sucrose) for2×20 second bursts. The homogenate was then transferred into a steriletube and spun at 4° C. (2000 rpm; for 5 min). The clarified homogenatewas transferred to a chilled cryovial, snap frozen and stored at −80° C.The supernatants from lung homogenates were used for the plaque assay.

HEp2 cells were grown in 24-well plates (Corning) for 48 hr prior toinfection in DMEM containing 10% FBS until they attained 100%confluency. Lung homogenate was thawed at RT and ten-fold serialdilutions were prepared in serum-free DMEM. The growth medium from HEp2cells was aspirated and replaced with 300 μL of serially diluted lunghomogenate and left to infect (4 h; 37° C./5% CO₂). The infectious mediawere aspirated and replaced with Plaque Assay Overlay (500 μL; 1%methylcellulose in MEM, 2% FBS, 1% Pen Strep, 0.5 μg/mL amphotericin B),and left for 7 days (37° C./5% CO₂). Cells were fixed with ice-coldmethanol for 10 min and blocked with 5% powdered milk (Marvel) in 0.05%PBS-tween (blocking buffer) for one hr at RT.

Anti-RSV F-protein antibody [2F7] (Abcam: ab43812) was diluted to a1:100 concentration in blocking buffer and added to cells for 1 hr at RTwith shaking. Cells were washed using PBS and then incubated with thesecondary antibody (HRP conjugated goat anti-mouse secondary antibody(Dako P044701-2) diluted in 1:400 with blocking buffer) for 1 hr at RTwith shaking. The secondary antibody solution was removed and cells werewashed with PBS before the metal-enhanced development substrate DAB wasprepared in ultra-pure water (according to manufacturer's instructions).Each well received 300 μL of development substrate (sigmaFAST D0426)until plaques were visible. Plaques were counted by eye and confirmedusing light microscopy, allowing the calculation of plaque forming unitsper mL of lung homogenate supernatant.

RSV Infection in Cotton Rats

Male Sigmodon hispidus cotton rats between 6 and 8 weeks of age wereinfected with hRSV/A/Long (ATCC, Manassas, Va.; 10⁵ pfu) in a volume of0.1 mL of sucrose stabilizing media. Compound (I) was dissolved in 100%DMSO (at 3.3, 10, 33 and 100 mg/mL), then diluted at 1:10 in isotonicsaline to achieve 10% DMSO in all treatments. Formulations were thensonicated to produce suspensions. The resulting suspensions wereadministered intranasally (50 μL) by pipette 4 hr before infection (onday 0), and then on days 1, 2 and 3 post infection. Four days after RSVchallenge, the animals were euthanised and the lungs were removed. Theleft lobe was used for viral titration via plaque assay and the lingularlobe for RSV/A/Long NS-1 qRT-PCR and cytokine qRT-PCR.

The supernatant of lung homogenates were diluted 1:10 and 1:100 in Eagle(E)-MEM. Confluent HEp-2 monolayers in 24-well plates were infected induplicate (50 μL of sample per well) starting with undiluted (neat)samples followed by diluted homogenates. After incubation for 1 hr (37°C./5% CO₂) wells were overlaid with 0.75% methylcellulose medium andplates replaced in the 37° C. incubator. After incubation (for 4 days),the overlay was removed, the cells fixed with 0.1% crystal violet stain(for 1 hr) and then rinsed and air-dried. Plaques were counted and viraltiters were expressed as plaque forming units per gram (pfu·g⁻¹) oftissue.

Total RNA was also extracted from homogenized lung tissue (RNeasypurification kit; Qiagen) and a sample (1 μg) was used to prepare cDNAusing QuantiTect Reverse Transcription Kit (Qiagen). For real-time PCRreactions (RSV NS-1 and RANTES genes) the QuantiFast SYBR® Green PCR Kit(Qiagen) was used in a final volume of 25 μL, with final primerconcentrations of 0.5 μM. Amplifications were performed on a Bio-RadiCycler for 1 cycle of 95° C. for 3 min, followed by 40 cycles of 95° C.for 10 sec, 60° C. for 10 sec, and 72° C. for 15 sec. Baseline cyclesand cycle threshold (Ct) were calculated by the iQ5 software in the PCRBase Line Subtracted Curve Fit mode. The standard curves were developedusing serially diluted cDNA sample most enriched in the transcript ofinterest (e.g., lungs from day 4 post-primary RSV infection). The Ctvalues were plotted against logo cDNA dilution factor. These curves wereused to convert the Ct values obtained for different samples to relativeexpression units which were then normalized to the level of β-actin mRNA(“housekeeping gene”) expressed in the corresponding sample. The mRNAlevels were expressed as the geometric mean±SEM for all animals in agroup.

In Vitro Screening Results

The profile of Compound (I), as disclosed herein, is summarised below(Table A) and demonstrates potent inhibitory activities against both RSVA2-induced CPE and RSV B-induced CPE in HEp2 cells. Furthermore, thecompound of the invention exhibits potent inhibition of RSV A2 F-proteinexpression in BEAS2B bronchial epithelial cells. No effect on cellviability, resulting from incubation with Compound (I), was detected.

TABLE A The effects of treatment with Compound (I) on RSV A2- and RSVB-induced CPE in HEp2 cells, on RSV A2 F-protein expression in BEAS2Bbronchial epithelial cells and on cell viability. IC₅₀/CC₅₀ Values (nM)or Inhibition (%) at indicated concentration RSV A2 RSV B RSV A2 CellCPE CPE F-protein Viability IC₅₀ % IC₅₀ % IC₅₀ CC₅₀ Treatment (nM)Inhibition¹ (nM) Inhibition² (nM) (nM) Compound 0.017 100 10.2 720.17 >14000 (I) Table Footnotes: ¹Inhibition (%) at 0.1 μg/mL;²Inhibition (%) at 1 μg/mL;

Anti-viral effects were also evaluated using air-liquid cultured humanprimary bronchial epithelial cells. The cells undergo extensivemucociliary differentiation, resulting in cultures with morphologicalcharacteristics similar to those observed in the normal humanrespiratory epithelium. As a result, this cell model closely mimics RSVinfections in human airways.

The RSV titre increased from day 1, peaked at day 3 and then graduallyand moderately reduced up to day 7. Treatment with Compound (I) to anapical well daily from day 0 to day 7 (early intervention, see Table B,FIG. 1) induced concentration dependent inhibition, and showed completeinhibition at 0.1 μg/mL over 7 days. Treatment with Compound (I) alsoproduced a dramatic reduction of virus titre on days 6 and 7 postinfection when it was administered from day 3 after the virus peak (Lateintervention, see Table C, FIG. 2).

TABLE B The effects of early intervention (days 0-7) with Compound (I)on RSV A2 viral titre in apical wash from RSV A2 infected, air-liquidinterface cultured, bronchial epithelial cells. Drug Virus titre inapical wash on days indicated Conc. expressed as the geometric mean (logPFU/mL) ± SD^(1,2) Treatment mg/mL 0 1 2 3 4 5 6 7 Vehicle none 0.0 ±0.0 0.94 ± 1.3   4.0 ± 0.18  4.5 ± 0.15  3.7 ± 0.22  4.1 ± 0.22  4.1 ±0.22  3.5 ± 0.31 plus virus Compound 0.004 0.0 ± 0.0 0.61 ± 0.86  3.1 ±0.22  3.9 ± 0.18  4.1 ± 0.16  4.2 ± 0.12  3.2 ± 0.26  2.8 ± 0.43 (I)plus 0.02 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 1.4 ± 1.1 1.9 ± 1.40.0 ± 0.0 0.0 ± 0.0 virus 0.1 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.00.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 ¹Allocated 1 PFU/mL if anyplaque was detected in the assay with ×10 diluted apical wash; ²The nvalues were 3 for all experiments.

TABLE C The effects of late intervention (days 3-7) with Compound (I) onRSV A2 viral titre in apical wash from RSV A2 infected air-liquidinterface cultured bronchial epithelial cells. Drug Virus titre inapical wash on days indicated Conc. expressed as the geometric mean (logPFU/mL) ± SD^(1,2) Treatment mg/mL 0 1 2 3 4 5 6 7 Vehicle none 0.0 ±0.0 2.4 ± 0.26 4.2 ± 0.12 5.2 ± 0.18 4.0 ± 0.14 4.2 ± 0.17  4.0 ± 0.503.5 ± 0.22 plus virus Compound (I) 0.02 0.0 ± 0.0 1.6 ± 1.1  3.9 ± 0.155.2 ± 0.04 3.8 ± 0.50 3.6 ± 0.13 0.0 ± 0.0 1.3 ± 0.94 plus virus 0.1 0.0± 0.0 2.3 ± 0.35 4.1 ± 0.14 5.0 ± 0.14 3.8 ± 0.31 1.7 ± 1.2  0.0 ± 0.00.0 ± 0.0  ¹Allocated 1 PFU/mL if any plaque was detected in the assaywith ×10 diluted apical wash; ²The n values were 3 for all experiments.In Vivo Testing

Human RSV is able to infect and replicate in a number of animal speciesused for pre-clinical screening, thereby enabling the performance andprofiles of novel anti-infective agents to be assessed and compared invivo (Bem, et al., 2011). Although primate species can also be infectedand studied, most work of this nature is conducted in mice or cottonrats. Both standard, inbred mouse strains and cotton rats arecharacterised as “semi-permissive” for the replication of human RSV,although significantly greater viral replication is seen in cotton ratscompared to inbred mouse strains. Compound (I) was therefore tested inthe above mentioned in vivo systems.

In RSV A2 infected mice, virus titre peaked on day 4 followinginoculation. Compound (I) was administered 1 day and 1 hr beforeinoculation (day 0) and then 2 and 3 days after virus infection eitherintranasally (Table D, FIG. 3) or intratracheally (Table E) and in bothcases demonstrated potent dose-dependent inhibition of viral titre inlung homogenates.

TABLE D The effects of intranasal treatment with Compound (I) on RSV A2viral titre in lung from RSV A2 infected mice. Drug Virus titre (logPFU/lung)¹ Conc Geometric Interquartile Treatment (mg/mL) mean Medianrange Vehicle plus none 3.0 3.2 2.8-3.4 virus Compound 0.2 2.4 2.51.9-3.0 (I) plus virus 2 <1.5² <1.5² ¹n values were 8 for allexperiments; ²Lower limit of quantitation (LOQ).

TABLE E The effects of intratracheal treatment with Compound (I) on RSVA2 viral titre in lung from RSV A2 infected mice. Virus titre (logPFU/lung)¹ Drug Conc Geometric Interquartile Treatment (mg/mL) meanMedian range Vehicle plus none 3.1 3.1 2.7-3.4 virus Compound 0.2 <1.5²<1.5² (I) plus virus ¹n values were 8 for all experiments; ²Lower limitof quantitation (LOQ).

The potent dose-dependent inhibition of virus titre by Compound (I) wasalso seen in lung homogenates from RSV/S/Long infected cotton rats onday 4 (Table F, FIG. 4). In addition, the drug substance displayed adose-dependent inhibition of RSV NS-1 gene transcripts (Table G) and ofRANTES transcripts in lung (Table H).

TABLE F The effects of intranasal treatment with Compound (I) on RSV A2viral titre in lung from RSV A2 infected cotton rats. Virus titre (logPFU/lung)¹ Drug Conc Geometric Interquartile Treatment (mg/mL) meanMedian range Vehicle none <2.3² <2.3² — Vehicle none 5.0 5.1 4.8-5.2plus virus Compound 0.33 4.7 4.7 4.5-4.8 (I) 1.0 4.5 4.5 4.4-4.8 plus3.3 <2.3² <2.3² — virus 10 <2.3² <2.3² — ¹n values were 6 for allexperiments; ²Lower limit of quantitation (LOQ).

TABLE G The effects of intranasal treatment with Compound (I) on RSV A2NS-1 gene expression in lung from RSV A2 infected cotton rats. RSV NSIgene transcript (/β-actin)¹ Drug Conc Interquartile % Treatment (mg/mL)Median range inhibition Vehicle none 0 0-0 — Vehicle none 4.4 2.4-6.4 —plus virus Compound 0.33 2.0 1.7-2.5 55% (I) 1.0 1.6 1.2-3.0 64% plus3.3 1.0 0.33-2.4  77% virus 10 1.0 0.36-2.2  77% ¹n values were 6 forall experiments.

TABLE H The Effects of intranasal treatment with Compound (I) on RANTESgene expression in lung from RSV A2 infected cotton rats. RANTES genetranscript (/β-actin)¹ Drug Conc Interquartile % Treatment (mg/mL)Median range inhibition Vehicle none 0.088 0.046-0.090 — Vehicle none0.29 0.21-0.40 — plus virus Compound 0.33 0.21 0.16-0.33 28 (I) 1.0 0.160.15-0.25 45 plus 3.3 0.11 0.075-0.15  62 virus 10 0.13 0.13-0.17 55 ¹nvalues were 6 for all experiments.

SUMMARY

The in vitro antiviral activity of the compound of the invention hasbeen demonstrated by its cytoprotective effect on HEp2 cells, infectedwith RSV. In this assay system the inhibition of virus replication wasdetected and quantified from the resulting inhibition of virus-mediatedCPE. It is particularly noteworthy that Compound (I) is a potentinhibitor of the CPE induced by both the RSV A strain the RSV B strainstudied. The potent antiviral activity of Compound (I) was furtherevidenced by its inhibition of RSV A2 F-protein expression in BEAS2Bcells.

The compound of the invention demonstrates low mammalian cell toxicityas measured by its lack of any significant effect in the cell viabilityassay. Furthermore, in an in vitro model of human lung epithelium,comprising an air-liquid interface culture of bronchial epithelialcells, the compound of the invention completely inhibited virus titrewhen administered by either early or late stage intervention. The latterobservation is particularly significant for the treatment of establisheddisease.

The in vivo antiviral activity of the compound of the invention has beendemonstrated in mice and cotton rats infected with RSV. In the assaysystems the inhibition of virus replication was detected and quantifiedfrom the RSV titre in lung homogenates as measured in a plaque assay. Inkeeping with the data obtained from the studies conducted inALI-cultured human bronchial cells, Compound (I) completely inhibitedvirus titre in the lungs of RSV A2 infected mice and cotton rats. Thecompound of the invention thus has the necessary attributes to be aneffective medicine for the treatment and/or prevention of RSV infectionand associated disease.

REFERENCES

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Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

All patents and patent applications referred to herein are incorporatedby reference in their entirety.

The invention claimed is:
 1. A compound of formula (I),

or a pharmaceutically acceptable salt thereof.
 2. A method of treatmentof RSV infection or disease associated with RSV infection in a subjectwhich comprises administering to said subject an effective amount of thecompound according to claim
 1. 3. A method of prevention of RSVinfection or disease associated with RSV infection in a subject whichcomprises administering to said subject prior to infection an effectiveamount of the compound according to claim
 1. 4. A method according toclaim 2 wherein the RSV infection is infection by viruses of the RSV Astrain and/or viruses of the RSV B strain.
 5. A method according toclaim 2 wherein the treatment is in combination with a second or furtheractive ingredient.
 6. A pharmaceutical composition comprising thecompound according to claim 1 optionally in combination with one or morepharmaceutically acceptable diluents or carriers.
 7. A pharmaceuticalcomposition according to claim 6 comprising the compound of formula (I)in particulate form suspended in an aqueous medium.
 8. A pharmaceuticalcomposition according to claim 6 which comprises a second or furtheractive ingredient.
 9. A pharmaceutical composition according to claim 8wherein the second or further active ingredient is selected fromanti-viral agents and anti-inflammatory agents.
 10. A compositionaccording to claim 9 wherein the second or further active ingredient isselected from F protein inhibitors, RNA polymerase inhibitors andribavirin.
 11. A method according to claim 3 wherein the RSV infectionis infection by viruses of the RSV A strain and/or viruses of the RSV Bstrain.
 12. A method according to claim 4 wherein the RSV infection isinfection by viruses of the RSV A strain.
 13. A method according toclaim 11 wherein the RSV infection is infection by viruses of the RSV Astrain.